Project 2 Molecular signatures for ME/CFS sub-types Abstract We propose the most comprehensive project to date to understand the metabolic and transcriptional perturbations that occur in ME/CFS. The investigators have extensive expertise in metabolomics and functional genomics, and access to banked samples from well-characterized cohorts of ME/CFS and control individuals. We will also have access, through Project 3, to samples obtained before and after orthostatic challenge and exercise. There are some intriguing indications from prior, limited studies that suggest that both metabolomic and transcriptomic approaches could yield insights into this poorly understood disorder. Transcriptional studies in ME/CFS performed on circulating leukocytes have indicated a combination of immune cell dysfunction and altered metabolic properties, but have generally been performed on limited numbers of individuals using less advanced transcriptional profiling than we propose in the current project. Published metabolomic studies in ME/CFS have consistently revealed evidence for abnormalities, with evidence for disturbances in lipid metabolism and neurotransmitter-related pathways in particular. Project Co-Lead Fiehn has generated preliminary data for 50 ME/CFS cases and 50 controls, showing results that support lipid and neurotransmitter metabolism abnormalities, in particular involving complex lipids and tryptophan metabolites. Co-Lead Greally?s group will study the same patients as Fiehn, following up on prior published studies with a focus on peripheral blood mononuclear cell (PBMC) transcriptional patterns. The Greally group has developed an approach that uses single cell transcriptomic reference data in combination with a published analytical algorithm to measure the proportions of cell subtypes in PBMCs from RNA-seq data. This allows a gene expression change in PBMCs to be attributed to either a change in cell subtype proportion, or to a genuine alteration in expression of a gene. This allows, for the first time, two distinct but individually interesting biological events to be distinguished ? the cell subtype and transcriptional alterations associated with ME/CFS. Our analytical focus is on the integration of the metabolomic and the transcriptomic information in Project 2, but we will also explore the associations with the clinical and laboratory data generated in the other projects, allowing us to link with microbiome and clinical variability. We note that the epigenetics field has recently begun to link both high body mass index and hyperlipidemia with changes in DNA methylation of peripheral blood leukocytes, with the DNA methylation found to be induced by both obesity and high lipid levels. This represented an unexpected reverse causation outcome that links metabolomic and transcriptional regulatory mechanisms, and serves as a foundation for the linked studies of this Project. Our two groups have extensive expertise in metabolomic and functional genomic research, which we will now be focusing on this extremely difficult disorder, but with early results yielding insights that indicate the promise of these molecular approaches in understanding and finding interventions to treat individuals with ME/CFS.